Patients with severe hemophilia A, after therapeutic exposure to factor VIII (fVIII), may develop inhibitory antibodies (Ab) to fVIII, that make their treatment difficult and costly. Antigen (Ag)-specific tolerance can be induced by administering the Ag through routes [e.g. nasal subcutaneous (s.c.)] that stimulate an immune response, than to abrogate an established response. In hemophilia patients with inhibitors, prevention of their further inhibitory response to forms of fVIII antigenically different from human fVIII may be easier to obtain that abrogation of their existing response to human fVIII. Porcine fVIII (pfVIII) is a possible such alternative to human fVIII. Mice genetically deficient in fVIII are a good model of hemophilia A, including appearance of inhibitors after intravenous (i.v.) Exposure to fVIII. To determine whether tolerization procedures using synthetic epitopes recognized by pfVIII-specific CD4+ cells prevent an immune response to pfVIII, in hemophilia A mice which already had inhibitors to human fVIII, will help development of similar treatments for hemophilia patients. The specific aims will be: 1) To determine the epitopes recognized and the cytokines secreted by anti- fVIII CD4+ cells in hemophilia A mice immunized with pfVIII, using overlapping peptides spanning the pfVIII sequence. To know the epitope repertoire of anti-pfVIII CD4+ cells, and whether they are of the Th1, Th2 or other subsets will help selecting the best tolerization procedures. 2) To determine whether prior i.v. exposure of hemophilia A mice to human fVIII affects the epitopes recognized by anti-pfVIII CD4+ cells after i.v. administrations of pfVIII. 3) To use synthetic pfVIII peptides forming CD4+ epitope sequences, for tolerization procedures in hemophilia A mice. Nasal tolerization procedures will be attempted first, using synthetic pfVIII CD4+ epitopes. If nasal tolerization will not prevent development of CD4+ and Ab responses to pfVIII, s.c. OR I.V. tolerization procedures will be attempted, using the same pfVIII epitope peptides. 4) To investigate the epitope repertoire of anti-pfVIII CD4+ cells in hemophilia patients treated sequentially with human and porcine fVIII to assess whether the characteristics of the anti-fVIII CD4+ cells in the mice are representative of those of CD4+ cells in hemophilia patients treated with human and porcine fVIII.